Mass spectrometry is a technique that weighs individual molecules, thus providing valuable chemical information. A mass spectrometer operates by exerting forces on charged particles (ions) in a vacuum using magnetic and electric fields. A compound must be charged (ionized) to be analyzed in a mass spectrometer. The ions must be introduced in the gas phase into the vacuum of the mass spectrometer. Ionizing large molecules of biological origins such as proteins, peptides and strands of DNA and RNA has proven difficult in the past since these molecules have effectively zero vapour pressure and are labile. A major thrust in mass spectrometry for some time has been the development of ionization sources for such large bio-molecules.
With the mapping of the genome, much research is now focused on understanding how cells function, individually and as a component in a tissue or a larger organism. It is hoped that this information will be useful for the control and eradication of certain diseases and the repair of damaged body parts. It is believed that the characterization and measurement of proteins expressed in cells will enhance the understanding of cellular function. A challenge in protein measurement, however, is sensitivity since there are estimated to be approximately 100,000 distinctly different proteins in any one cell. There could be as few as one or two proteins in any one cell or as many as several hundred or more. Currently, the only way to study the expression levels of proteins is to isolate a population of cells, typically more than 1 million cells, and perform analysis on the proteins isolated from that population of cells. Even in these situations, however, the proteins that are expressed at low levels are generally not identified because their numbers are below the level of detection.
Electrospray ionization (“ESI”) and matrix-assisted laser desorption and ionization (“MALDI”) are two techniques that have been developed to ionize large bio-molecules.
ESI is a desolvation method in which a high DC electric potential is applied to a metallic capillary needle that is separated from a counter electrode held at a lower DC potential. The electric field causes a liquid (containing the analyte in solution) emerging from the capillary to be dispersed into a fine spray of millions of charged droplets. The droplets in the aerosol carry a net charge of the same polarity as the electric field. As the solvent evaporates from the droplets, the droplets decrease in size, increasing the charge concentration on the droplet surface. Eventually, a “Coulombic explosion” occurs when Coulombic repulsion overcomes a droplet's surface tension. This results in the droplet exploding, forming a series of smaller, lower charged droplets. This process of shrinking and exploding repeats until individually charged analyte ions are formed. The rate of solvent evaporation can be increased by introducing a drying gas flow counter to the current of the sprayed ions. Nitrogen is frequently used as the drying gas.
With evaporation of the solvent from the droplets, the cyclical process of coulomb fission and solvent evaporation ultimately leads to the deposition of net charge onto the analyte molecule (e.g. blo-molecule) in the droplet. The bio-molecule, adducted by, for example, multiple protons, is desorbed from the droplet at atmospheric pressure. A small fraction of these ions pass through an orifice into the vacuum of the mass spectrometer for analysis.
A disadvantage of the ESI method is that only a small fraction (0.01% or less) of the sample material is utilized. The majority of the material emerging from the capillary ends up on the counter electrode or on the plate that has the sampling orifice. The reason for this is that the electric field that disperses the liquid solution into droplets is also responsible for causing detrimental space charge effects. Space charge effects arise because each droplet, and the resulting ions in the aerosol plume, all carry net charge of the same polarity, causing these droplets/ions to repel one another because of electrostatic repulsion. This causes the spray of droplets leaving the tip of the capillary to spread out into a cone having its apex at the tip of the capillary. Hence, the overall sample utilization efficiency is low in conventional ESI methods because the droplets/ions at atmospheric pressure are extremely difficult to focus through the sampling orifice. This limits the effectiveness of ESI if only a small amount of analyte is available for analysis, which is often the case in respect of bio-molecules.
MALDI involves the deposition of a sample, usually as a liquid, onto a flat plate or into recessed wells formed in a plate. A matrix of one or more compounds is also used. The matrix may be a solid or a liquid. The sample material can be deposited as a layer on top of or below the matrix or intimately mixed with the matrix. Typically, the matrix molecules are present in the starting solution in a concentration approximately 1000 times greater than the analyte molecules. After deposition, the plate is exposed to a pulsed laser beam. The matrix absorbs the energy from the laser, causing rapid vibrational excitation and desorption of the chromophore. The matrix molecules evaporate away and the desorbed analyte molecules can be cationized by a proton or an alkali metal ion. The ionized analyte molecules can be analyzed using a time-of-flight (“TOF”) analyzer. In such a case, the overall technique is often referred to as matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (“MALDI-TOF-MS”).
Small sample spots produce higher sensitivity in MALDI. It has been suggested that the current fundamental limit for MALDI is 5 molecules per μm2 and that providing a method of creating spots of a sample that are only 1-5 μm in diameter will lower the detection limit for MALDI: Keller, B. O. and Li, L. J. Am. Soc. Mass Spectrum. 2001, 12, 1055-1063. This could be accomplished using smaller capillary sizes to create smaller droplets. As has been pointed out, however, handling of volumes of picoliters becomes problematic in smaller inner diameter capillaries because of the higher surface to volume ratio that leads to stronger tension forces.
The need has therefore arisen for a method and apparatus for producing a source of ions, suitable for mass spectrometric analysis, from a discrete particle. The need has also arisen for improved techniques for depositing an analyte, such as a bio-molecule, onto a plate for MALDI mass spectrometry.